Whole blood is made up of various cellular components such as red cells, white cells and platelets suspended in its liquid component, plasma. Whole blood can be separated into its constituent components (cellular or liquid), and the desired separated component can be administered to a patient in need of that particular component. For example, platelets can be removed from the whole blood of a healthy donor, collected, and later administered to a cancer patient, whose ability to “make” platelets has been compromised by chemotherapy or radiation treatment.
Commonly, platelets are collected by introducing whole blood into a centrifuge chamber wherein the whole blood is separated into its constituent components, including platelets, based on the size and densities of the different components. This requires that the whole blood be passed through a centrifuge after it is withdrawn from, and before it is returned to, the donor. Typical blood processing systems thus include a permanent, reusable centrifuge assembly containing the hardware (drive system, pumps, valve actuators, programmable controller, and the like) that spins and pumps the blood, and a disposable, sealed and sterile fluid processing assembly that is mounted cooperatively on the hardware. The centrifuge assembly spins a disposable centrifuge chamber in the fluid processing assembly during a collection procedure, thereby separating the blood into its constituent components.
“On line” automated blood separation systems are today used to collect large numbers of platelets. On line systems perform the separation steps necessary to separate platelets from whole blood in a sequential process with the donor present. On line systems draw whole blood from the donor, separate out the desired platelets from the drawn blood, and return the remaining red blood cells and plasma to the donor, all in a sequential flow loop. Large volumes of whole blood can be processed using an automated on line system. Due to the large processing volumes, large yields of concentrated platelets can be collected. Moreover, since the donor's red blood cells are returned, the donor can donate platelets for on line processing much more frequently.
In the automated, on-line separation and collection of platelets, sometimes referred to as platelet apheresis or simply “plateletpheresis”, the platelets are separated from whole blood and concentrated in the centrifuge chamber or elsewhere in the fluid processing set (hereinafter “platelet concentrate” or “PC”). Although most of the plasma is removed during apheresis, a volume of plasma still remains in the PC, hereinafter referred to as “residual plasma”. Platelets may also be derived from buffy coats obtained from manually collected units of whole blood. A plurality of buffy coats are typically pooled to provide an amount or dose of platelets suitable for transfusion. The platelets, whether collected by apheresis or from pooled buffy coats, are typically reconstituted in a liquid medium, such as plasma and/or a synthetic storage solution, for storage until needed for transfusion to a patient.
For the stored platelets to be suitable for later administration they must substantially retain their viability and platelet function. A number of interrelated factors may affect platelet viability and function during storage. Some of these factors include the anticoagulant used for blood collection, the method used to prepare the platelets, the type of storage container used, and the medium in which the platelets are stored.
Currently, platelets may be stored for five or even seven days at 22° C. After seven days, however, platelet function may become impaired. In addition to storage time, other storage conditions have been shown to affect platelet metabolism and function including pH, storage temperature, total platelet count, plasma volume, agitation during storage and platelet concentration.
A variety of assays have been developed which attempt to determine the quality of stored platelets and the in vivo viability of those platelets when transfused to a patient. For instance, the percentage of platelets that respond appropriately to an ADP agonist (the ESC assay) and the percentage of platelets that respond appropriately to hypotonic shock (HSR assay) are two assays which are thought to correlate well with viability of stored platelets. ESC is a photometric assessment of discoid platelet shape change in response to an ADP agonist. VandenBroeke, et al., “Platelet storage solution effects on the accuracy of laboratory tests for platelet function: a multi-laboratory study.” Vox Sanguinis (2004) 86, 183-188.
The results of the HSR (Hypotonic Shock Response) assay are often considered to correlate strongly with the in vivo effectiveness of platelets when they are introduced into an individual. This assay measures the ability of platelets to recover a discoid shape after swelling in response to a hypotonic environment. Higher scores on either the HSR or ESC assay appear to correlate with increased viability of the platelets when transfused to patients. The methods and uses of the HSR and ESC assays are described in more detail by Holme et al. Transfusion, January 1998; 38:31-40, which is incorporated by reference herein.
Another assay for measuring platelet viability based on platelet shape is the Morphology Score for the platelets during and after storage. Morphology Scores may be determined by, for example, the Kunicki Method, whereby a selected number of platelets in a sample are examined to determine the shape, e.g., discoid, spherical, dendritic or balloon. The number of each shape is then multiplied by a selected multiplier and the resultant numbers are summed to arrive at a Morphology Score. A score of 250-400 is typically indicative of a viable platelet population.
Another shape based assay is the so called “swirling assay” which has also been used as a measure of the quality of platelet concentrates. The swirling assay is based on the ability of discoid platelets to reflect light, producing a shimmering phenomenon. As described by Bertolini and Murphy, Transfusion 1994; 34:796-801 and Transfusion 1996: 36:128-132 and incorporated herein by reference, platelet samples scoring positive in a swirling assay are believed to be of higher quality than samples scoring intermediate or negative for swirling.
The presence of the glycoprotein P-selectin on the surface of platelets is also used to characterize the viability of platelets upon transfusion with the presence of P-selectin believed to indicate a loss of viability. As described by Holme et al. Transfusion 1997; 37:12-17 and incorporated herein by reference, platelets undergo a shape change transforming from disc shaped to sphere shaped upon platelet activation. This activation is thought to involve the secretion of β-thromboglobulin from the alpha granules resulting in the appearance of P-selectin on the surface of the platelets. Antibodies directed against P-selectin, such as the monoclonal antibody CD62P, are used to detect the presence of P-selectin on the surface of platelets and have been used as a marker of platelet activation and a decreased viability of the platelets upon transfusion.
Another marker of the quality of platelets is extracellular levels of lactate dehydrogenase. Lactate dehydrogenase is an intracellular enzyme and therefore higher extracellular levels of lactate dehydrogenase are thought to reflect increased levels of platelet lysis.
In order to maintain viability, platelets must generate new adenosine triphosphate (ATP) continuously to meet their energy needs. As shown in FIG. 1 platelets use two metabolic pathways to generate ATP: (a) anaerobic glycolysis followed by lactic acid fermentation or (b) glycolysis followed by oxidative phosphorylation. Glycolysis results in one mole of glucose being converted to 2 moles of pyruvate, and two moles of ATP. The pyruvate can then undergo lactic acid fermentation also called anaerobic glycolysis. Although no additional ATP is produced in lactic acid fermentation, the conversion of pyruvate to lactic acid regenerates NAD+ and allows glycolysis to continue generating at least a small amount of ATP from the metabolism of glucose. Because lactic acid fermentation, which can negatively affect the pH of the medium and platelets stored therein, is stimulated by the absence of oxygen, platelets are typically stored in containers permeable to oxygen to promote oxidative phosphorylation and suppress lactic acid formation.
In oxidative phosphorylation, pyruvate, fatty acid or amino acids are converted to CO2 and water in the citric acid cycle. This pathway requires the presence of an adequate supply of oxygen. Glycolysis followed by oxidative phosphorylation produces 36 moles of ATP per mole of glucose and therefore is much more efficient than glycolysis followed by lactic acid fermentation.
However, rather than utilizing oxidative phosphorylation exclusively, the platelets continue to produce lactic acid through anaerobic glycolysis. Therefore, even in the presence of adequate amounts of oxygen and when stored in media containing glucose, (media such as plasma and certain synthetic storage solutions) the utilization by platelets of glycolysis coupled with lactic acid fermentation for energy production results in the concentration of lactic acid increasing over time. As noted above, the increase in lactic acid gradually acidifies the storage media. This acidification of the media alters platelet physiology and morphology such that when the pH of the media drops below about 6 the platelets may be considered nonviable. Even drops in pH that are too small to render platelets nonviable have been observed to cause decreases in the total amount of ATP. These reductions in ATP affect platelet function as ATP plays a role in platelet adhesion and platelet aggregation. Consequently, it would be desirable to provide a storage medium for platelets that results in the prevention and/or delay of this decrease in pH.
A number of approaches for the storage of platelets for transfusion have been described. Although plasma is effective for storage of platelets, it may not be the ideal medium for platelet storage because plasma itself is a valuable blood component that can be removed from the platelets and then used or further processed for use in the treatment of patients with other disorders.
Another reason for removing some, most, or all of the plasma from platelet concentrate is to prevent allergic transfusion reactions (ATR) to plasma. There may be other reasons for removing at least some or even most of the plasma from the platelets. For example, the presence of certain antibodies in plasma has been correlated with the occurrence of TRALI (transfusion-related acute lung injury) in some patients. Consequently, while residual plasma may be present to some degree in platelets obtained in an apheresis procedure, it may be desirable to reduce the amount of residual plasma in platelets. Preferably, residual plasma may be removed from platelet concentrate in a way that is cost effective, efficient, and which does not affect the in vivo viability of the platelets. Accordingly, it would be desirable to develop automated systems and methods to remove residual plasma from platelets to obtain a platelet concentrate such that upon reconstitution, the platelet product has a substantially reduced volume of plasma such as less than approximately 5% of the total platelet product volume, or even less.
Reductions of residual plasma to below 5% and even to and/or less than 1% plasma may be achieved by “washing” platelets or PC to remove residual plasma. Upon resuspension of washed platelets, the volume of plasma remaining in the resuspended PC may preferably be less than approximately 1% of the total washed platelet product volume. Accordingly, it would be desirable to develop automated systems and methods for washing platelet concentrate to obtain a washed platelet product having less than approximately 1% plasma (and up to approximately 0%).
In place of the removed residual plasma, platelets may be resuspended and stored in some small volume of residual plasma and a substantially greater volume of an alternate storage medium. Synthetic aqueous solutions have been developed to provide a suitable environment for stored platelets, which may be “stand alone” solutions or, as indicated above, used in combination with some amount of plasma. Reducing the amount of residual plasma in platelet concentrate and then combining the plasma-reduced platelet concentrate with a synthetic storage medium may have advantages, such as preserving the in vivo viability of platelets and achieving extended storage times of such platelets. Thus, it would be desirable to combine PC or platelets having a reduced residual plasma volume with synthetic aqueous solutions to obtain a platelet product for storage. InterSol®, a commercially available platelet storage medium is generally described in U.S. Pat. No. 5,908,742 which is incorporated herein in its entirety. InterSol® contains sodium citrate, sodium acetate, sodium phosphate and adjusted to isoosmolarity with sodium chloride. A typical formulation of InterSol® contains 21.5 mM (3.05 g/L) dibasic sodium phosphate anhydrous (Na2HPO4), 6.7 mM (1.05 g/L) monobasic sodium phosphate (NaH2PO4.2H2O), 10.8 mM (3.18 g/L) sodium citrate 2H2O, 32.5 mM (4.42 g/L) sodium acetate 3H2O, and 77.3 mM (4.52 g/L) sodium chloride. The InterSol® solution is approximately isoosmolar (about 300 mOsm/L) with platelets and plasma, and has a pH of approximately 7.2. In certain applications (such as, but not limited to, inactivation of pathogens in platelets) InterSol® may be used in combination with plasma ratio of InterSol®/plasma ratio approximately 70%/30% to 60%/40%. Phosphate buffer in InterSol® stabilizes the pH of the solution during platelet storage and increases glycolysis.
While InterSol® has worked satisfactorily in the preservation of blood platelets, further improvements to the storage time and in vivo viability of platelets would be desirable. For example, as noted above, it would be desirable to develop a platelet storage media that reduces platelet utilization of lactic acid fermentation and thus slows the drop in pH that typically occurs during platelet storage, thereby increasing viability of stored platelets to seven days or more, up to as many as 15 days, at 22° C. It would also be desirable to develop a synthetic storage media that allows for the storage of platelets in a reduced amount of plasma (such as less than approximately 30%, preferably less than 5%, and even more preferably less than 1%). It would also be desirable to provide a platelet storage media with a lower concentration of phosphate and a sufficient supply of nutrients to substantially meet the energy needs of the platelets during storage while maintaining a pH between about 6.4 and about 7.4. Finally, it would be desirable to provide an automated system that is programmed for and allows for the collection of platelets in a reduced amount of plasma.